The dopaminergic neurodegeneration in the substantia nigra pars compacta (SNpc) is responsible for the marked motor impairment observed in Parkinson's disease (PD). However, the molecular mechanisms underlying this are not completely understood. Since by the time of diagnosis, 50-70% of the dopaminergic neurons of the nigrostriatal pathway have already been degenerated, it is difficult to investigate the early-stage events of disease pathogenesis. Due to inaccessibility of the human brain to study initial pathogenic mechanisms of the disease, experimental models have been developed in an attempt to elucidate PD etiology and its progression. Nevertheless, PD models are a controversial issue in neuroscience research since it is challenging to mimic human neuronal complexity. Therefore, the lack of optimal models that recreate disease pathology is one of the causes of failure of clinical trials that have attempted to find new/better PD therapies. Taking this in consideration, the development of more suitable models is necessary to improve our knowledge regarding PD etiological mechanisms. Additionally, the understanding of the advantages and disadvantages of models already established would also be beneficial for PD research, which our group addressed by reviewing this subject. Considering this, we chose SH-SY5Y cells as a PD model for our studies. To investigate the initial stages of PD-induced neurodegeneration, our work focused in the role of cofilin-1, a protein involved in mitochondrial dysfunction caused by oxidant-induced-apoptosis, which are two pathogenic processes strongly related to PD. Hence, in the thesis, we aimed to validate the use of retinoic-acid-(RA)-differentiated SH-SY5Y cells as an in vitro model and use it to investigate the potential role of cofilin-1 in the initial molecular and cellular mechanisms of PD. Although SH-SY5Y cells are widely used in PD research, their major drawback is their lack of important neuronal features, such as low levels of proliferation and stellate morphology. On the other hand, SH-SY5Y cells can acquire a neuronal phenotype when treated with differentiation agents such as RA. Since several protocols have been described, the consequence of which may be the discrepancies observed among studies regarding neuronal and dopaminergic features. In Chapter I, we aimed to validate a RA-differentiation protocol for SH-SY5Y cells previously established by our research group, focusing upon characterization of neuronal features and its subsequent response to 6-hydroxydopamine (6-OHDA), a toxin widely used to induce dopaminergic degeneration. RA-differentiated SH-SY5Y cells have low proliferative rates, a pronounced neuronal morphology and high expression of genes related to synapse vesicle cycle, dopamine synthesis/degradation, and dopamine transporter (DAT). After exploring phenotypic differences between these two models, we verified that RA-differentiated cells were more sensitive to 6-OHDA toxicity than undifferentiated cells, which could be related to an increase of DAT immunocontent. Many lines of evidence have showed that DAT is responsible for 6-OHDA uptake in vivo. Once inside the neuron, 6-OHDA underwent auto-oxidation causing a significant increase in oxidative stress. However, toxin uptake is not an essential step in undifferentiated SH-SY5Y cells, as auto-oxidation occurs extracellularly. We showed here, for the first time, that RA-differentiated SH-SY5Y cells can mimic, at least in part, an important mechanism of the 6-OHDA-induced cell death found in previous in vivo studies. Hence, the cellular model established by our research group presents essential neuronal features, being a suitable model for PD research. In Chapter II, RA-differentiated SH-SY5Y cells were used as cellular model to investigate disease molecular mechanisms, focusing upon cofilin-1. Our previous data have shown that oxidation of non-phosphorylate (activated) cofilin-1 leads to mitochondrial dysfunction and cell death induced by apoptosis in tumour cells. Here we found that cofilin-1 played a role in early stages of neuronal apoptosis induced by 6-OHDA in our cellular model since cofilin-1 mitochondrial translocation precedes organelle dysfunction. Overexpression of wild type CFL1 resulted in increased sensitivity of SH-SY5Y cells to 6-OHDA-induced neuronal cell death. Furthermore, overexpression of non-oxidizable CFL1 containing Cys-to-Ala mutations (positions 39, 80 and 139) increased neuronal resistance to this toxin, suggesting that oxidation is an important step in 6-OHDA toxicity. Follow-up experiments were performed in order to evaluate clinically whether cofilin-1 pathway proteins content is altered in PD post mortem human brain. Our findings showed a significant decrease in p-cofilin-1/cofilin-1 ratio in PD patients, which indicates an increase in the amount of activated cofilin-1 available for oxidation. Moreover, through principal component analysis, the immunodetection of cofilin-1 pathway proteins were able to discriminate controls and PD individuals during the early-stage of neuropathological changings. Hence, we demonstrated, for the first time, a possible role for cofilin-1 in PD pathogenesis and its potential use as biomarker. Taken together, our data showed that RA-differentiated SH-SY5Y cells present terminally-differentiated dopaminergic neuron features, that are essential to mimic dopaminergic neurons. By using this cellular model and post mortem brain tissue, we also demonstrated a possible role for cofilin-1 in early steps of the neurodegeneration process found in PD, which it could impact drug and biomarker discovery researches.
| Identifer | oai:union.ndltd.org:IBICT/oai:lume56.ufrgs.br:10183/159503 |
| Date | January 2017 |
| Creators | Lopes, Fernanda Martins |
| Contributors | Klamt, Fabio, Parsons, Richard B. |
| Source Sets | IBICT Brazilian ETDs |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, instname:Universidade Federal do Rio Grande do Sul, instacron:UFRGS |
| Rights | info:eu-repo/semantics/openAccess |
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